An essential step during the life cycle of human immunodeficiency virus type 1 (HIV-1) and other retroviruses is integration of a double-stranded DNA copy of the viral genome into a host cell chromosome. Integration of retroviral DNA is catalyzed by the viral protein integrase. The process is non-specific but is not random, with some integration sites used at much greater frequencies than others. The site of integration has important consequences to both the invading virus and the susceptible host, and the mechanism that determines target site selection is not well understood. The long-term objective of this proposal is to further understand the mechanism of HIV-1 integration and the process that controls the selection of host DNA sites for integration, and to devise a method of directing integration into a predetermined site. The specific aims are (1) to examine the distribution and preference of HIV-1 integration sites in the human genome and identify viral factors and host conditions that affect target site selection, and (2) to study site-directed integration of retroviral DNA using integrase fused to a sequence-specific DNA-binding protein. In Aim 1, libraries of HIV-1 integration sites will be generated to detect viral or host factors that can influence target site selection and determine whether the alteration can cause significant biological outcomes. The hypothesis that HIV-1 integration into certain chromosomal sites can preferentially lead to latency, a major contributing factor to the inability of current treatments to eradicate the virus, will be tested. To expedite the analysis, a new high-throughput assay for determining integration sites will be developed. Aim 2 will analyze the efficiency and specificity of a new class of integrase fusion proteins to mediate site-directed integration in cultured cells. These fusion proteins consist ofHIV-1 integrase and a polydactyl zinc-finger E2C, a synthetic DNA-binding protein that specifically binds to an 18-bp sequence with high affinity. These synthetic proteins can also be modified to potentially recognize any desired sequences. A new strategy in which the HIV-1 integrase-donor DNA complex is delivered to a DNA site of interest through protein-protein interactions will also be tested. The availability of an efficient and high throughput assay for examining the distribution and preference of integration sites will yield a better understanding of target site selection during retroviral infection and provide a better assessment on the relative risk of insertional mutagenesis. Studies on site-directed integration using fusion proteins may lead to a new approach for inserting exogenous genes at specific sites, which will have a wide application as an experimental tool and improve the therapeutic application of current retrovirus-based vectors for gene delivery.